Methods and compounds for labeling DNA with xanthine and lower alkyl substituted xanthine derivatives and reagents for the in situ detection of chromosomes

ABSTRACT

This invention discloses methods and compounds for covalently linking xanthine or lower alkyl substituted derivatives of xanthine to DNA and the resulting xanthine or lower alkyl substituted xanthine derivative labeled DNA reagents. Reagents for the in situ detection of a chromosome or a region of a chromosome are disclosed. These reagents include a multiplicity of DNA sequences that are complementary to different portions of the chromosome or chromosome region to be detected. Multiple xanthine or lower alkyl substituted xanthine derivative labels are covalently linked to the DNA sequences. These xanthine or lower alkyl substituted xanthine derivative labeled reagents are contacted under hybridizing conditions with the chromosome or chromosome region of interest. Any binding of the reagent with the chromosome or chromosome region of interest may then be detected by immunological techniques.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods and compounds for labeling DNA. Thepresent invention also relates to the detection and identification ofchromosomes or regions of chromosomes by hybridization of a multiplicityof different chromosome-specific probes. In particular, this inventionrelates to in situ hybridization of these chromosome specific probes tochromosomes from disrupted cells that have been prepared so as to leavethe native chromosome structure essentially intact and to preserve thephysical relationships between different chromosomes, different portionsof the same chromosome or between chromosomes and other cellularstructures.

2. Summary of the Related Art

In situ hybridization techniques are well known in the art, and have avariety of applications for the detection of chromosomes or regions ofchromosomes. These applications include but are not limited to prenataldiagnosis, genetic mapping, somatic cell hybridization and the detectionof specific chromosomal genetic markers of malignant and other diseases.Hybridization is performed in situ between chromosomal target DNAsequences and probe sequences that have been modified so as to bedetectable by a variety of physical or chemical means afterhybridization. Preparation of target chromosomal DNA is performed so asto result in the minimum amount of disruption of chromosomal andcellular structures and to permit hybridization with the probe. Methodsfor preparation of chromosomal target DNA are well known in the art.(See for example Gall and Pardue, Proc. Natl. Acad. Sci. USA 64: 600(1969), John et al., Nature (London) 223: 582 (1969), Rudkin andStollar, Nature (London) 265: 472 (1977)).

A large variety of hybridization probes, comprised of DNA and RNAsequences, are known in the art.

Gall and Pardue, supra, disclose the use of complementary RNA (cRNA)probes labeled with tritium-containing nucleotides for the detection ofspecific sequences on lampbrush chromosomes.

John et al., supra, disclose the use of cRNA probes labeled with ¹²⁵ Ito detect DNA:RNA hybrids.

Bauman et al., Exp. Cell Res. 128: 485-490 (1980) discloses detection ofribosomal 5S genes on polytene chromosomes in D. hyaei afterhybridization to labeled 5S RNA.

Malcolm et al., Ann. Hum. Genet. 45: 135-142 (1981) disclose thedetection of the human B-globin gene on chromosome 11 after in situhybridization of normal human chromosomes with a labeled complementaryDNA (cDNA) probe.

Manuelidis et al., J. Cell Biol. 95: 619-625 (1982) disclose detectionof mouse satellite DNA sequences in mouse chromosomes afterhybridization with a labeled mouse satellite-specific DNA probe.

Landegent et al., Exp. Cell Res. 153: 61-72 (1984) disclose detection ofmouse chromosomes after hybridization with cloned satellite DNAsequences.

Landegent et al., Nature (London) 317: 175-177 (1985) disclose detectionof the human thyroglobulin gene at a specific chromosomal location afterhybridization with labeled cosmid clone DNA.

Hopman et al., Histochemistry 85: 1-4 (1986) disclose detection of humanchromosomes in mouse: human somatic cell hybrids after hybridizationwith labeled total human DNA and detection of mouse chromosomes afterhybridization with cloned mouse satellite DNA sequences.

Landegent et al., Hum Genet. 73: 354-357 (1986) disclose the detectionof a specific portion of human chromosome 4 after hybridization with acloned genomic DNA probe, which has been genetically linked to the genefor Huntington's disease.

Moyzis et al., Chromosoma (Berlin) 95: 375-386 (1987) disclose specificdetection of portions of human chromosomes 9 and 16 after hybridizationwith cloned human satellite DNA probes.

van Dekken and Bauman, Cytogenet. Cell Genet. 48: 188-9 (1988) disclosespecific detection of human chromosome 1 after hybridization withfluorescently labeled cloned probes specific for the repetitivesequences found at the centromere and telomere.

Emmerich et al., Exp. Cell Res. 181: 126-140 (1989) disclose specificdetection of human chromosomes 1 and 15 after hybridization with clonedprobes to tandemly repeated human DNA.

Pieters et al., Cytogenet. Cell Genet. 53: 15-19 (1990) disclosedetection of human chromosomes in individual sperm cells afterhybridization with labeled chromosome-specific probes.

van Dekken et al., Cytometry 11: 153-164 (1990) disclose detection byflow cytometry of chromosomes 1 and Y in interphase and metaphase nucleiof normal human blood cells after hybridization with chromosome-specificlabeled DNA probes.

Cremer et al., Cytometry 11: 110-118 (1990) disclose the detection ofspecific chromosomal damage induced by treatment of human peripherallymphocytes with ionizing radiation after hybridization with labeledchromosome-specific DNA probes.

Kievits et al., Cytometry 11: 105-109 (1990) disclose the detection inhuman lymphocyte preparations of specific chromosomes or portions ofchromosomes after hybridization with labeled total genomic DNA ofmouse:human somatic cell hybrids and after pretreatment of the labeledDNA with unlabeled total human genomic DNA.

Methods for labeling DNA and RNA probes are well known in the art. Thesemethods include enzymatic incorporation of modified nucleotides,chemical synthesis of probes containing modified nucleotides and directmodification of nucleotides. The modifications include derivatization ofnucleotides fort he addition of labels or haptens, and the directaddition of labels or haptens. Traditional methods involved theincorporation of radioactive isotopes such as ³ H, ¹⁴ C, ³⁵ S, ³² P and¹²⁵ I. The inherent instability and correspondingly short usefullifetime of probes incorporating radioactive label, as well as healthand waste management concerns, have prompted the adoption of a number ofnonradioactive compounds for labeling probe DNA and RNA.

U.S. Pat. No. 4,833,251 discloses direct and synthetic modification ofDNA probes, and teaches derivatization of DNA as an N 4! (substitutedamino) cytosine. This derivatization is taught by incorporation ofderivatized nucleotides into DNA synthesized enzymatically or bysolid-phase chemistry. Derivatization is also taught by directmodification of single stranded DNA prepared from genomic eukaryotic DNAand from DNA cloned in prokaryotes using techniques well known to theskilled.

U.S. Pat. No. 4,828,979 discloses biotin modification of nitrogen atomsat nucleotide bases, which are involved in Watson-Crick base pairing ofhybridized DNA and RNA sequences, and teaches the synthesis of probeslabeled with biotin at these positions.

U.S. Pat. No. 4,626,501 teaches the derivatization of DNA and RNAsequences at adenosine and cytosine residues by conjugation of theseresidues at nitrogen atom positions N-6 and N-4, respectively, with thecompound 3-(4-bromo-3-oxobutane 1-sulfonyl)-propionate and a number ofrelated compounds.

Dale et al., Biochemistry 14: 2447-2457 (1975) teach the derivatizationof nucleotides and polynucleotides with mercurated compounds.

Bauman et al., Exp. Cell Res. 128: 485-490 (1980) teach the chemicalconjugation of fluorescent compounds directly to the 3' hydroxyl moietyof RNA molecules after treatment with sodium periodate.

Langer et al., Proc. Natl. Acad. Sci. U.S.A. 78: 6633-6637 (1981)disclose the enzymatic incorporation into probe DNA and RNA of UTP anddUTP that has been modified at the carbon atom 5 (C-5) position of thenucleotide.

Renz, EMBO J. 2: 817-822 (1983) teaches the conjugation of singlestranded DNA probes with biotin or ¹²⁵ I labeled histone H1 protein.

Shroyer and Nakane, J. Cell Biol. 97: Abstract 377a (1983) disclose thederivatization of cDNA probes by treatment with dinitrophenol.

Tchen et al., Proc. Natl. Acad. Sci. USA 81: 3466-3470 (1984) discloseaddition of the hapten 2-acetylaminofluorene to single stranded anddouble stranded DNA and RNA probes specifically at guanosinenucleotides.

Landegent et al., Exp. Cell Res. 153: 61-72 (1984) teach the use of2-acetylaminofluorene labeled mouse satellite DNA probes for the in situdetection of mouse chromosomes.

Draper, Nucleic Acids Res. 12: 989-1002 (1984) teaches thederivatization of polynucleotides by bitsulfite-catalyzed transaminationand discloses the labeling of derivatized polynucleotides with afluorescent compound, nitrobenzofurazan.

Forster et al., Nucleic Acids Res. 13: 745-761 (1985) disclose labelingof DNA and RNA photochemically with a photoactive derivative of biotin.

Hopman et al., Histochemistry 85: 1-4 (1986) disclose the use ofmercurated human total genomic DNA probes for in situ detection of humanchromosomes.

A number of methods well known in the art have been developed for thedetection of nonradioactively labeled probes. These methods includedirect detection of fluorescently labeled compounds, and indirectmethods, which rely on the binding of a reporter molecule that is thendetected either directly or indirectly. These reporter-based methodsinclude immunological methods, in which antibodies recognize either thetarget:probe hybrid molecule itself or the hapten derivatized to theprobe, and affinity methods, which are based on specific interactionswith hapten molecules derivatized to the probe. Detection of thesereporter molecules has been achieved by the attachment of a fluorescentlabel, conjugation to an enzyme followed by enzymatic conversion of itssubstrate to a detectable product, or conjugation with electron-denseatoms, such as gold, iron or silver, and detection either visually or byelectron microscopy.

Manning et al., Chromosoma 53: 107-117 (1975) disclose that biotin,covalently attached to RNA via electrostatic interaction between RNA andcytochrome c, can be detected after in situ hybridization by binding offerritin-conjugated avidin molecules and visualization by electronmicroscopy.

Rudkin and Stollar, Nature (London) 317: 472-473 disclose detection ofin situ hybridization by the use of fluorescently labeled antibodiesagainst RNA:DNA hybrids.

Hutchinson et al., J. Cell Biol. 95: 609-618 (1982) disclose the use ofavidin conjugated with colloidal gold to detect in situ hybridization ofbiotinylated probes by electron microscopy.

Manuelidis et al., J. Cell Biol. 95: 619-625 (1982) disclose the use offluorescently labeled antibodies to detect in situ hybridization withbiotinylated mouse satellite DNA probes.

Landegent et al., Hum. Genet. 73: 354-357 (1986) disclose the use ofhorseradish peroxidase-conjugated antibodies to detect in situhybridization of a 2-acetylaminofluorene-conjugated cosmid probe for aHuntington's disease gene.

Hopman et al., Histochemistry 84: 169-178 (1986) disclose the use ofsulfhydryl haptens to mercurated DNA probes for the immunologicaldetection of in situ hybridization.

Garson et al., Nucleic Acids Res. 15: 4761-4770 (1987) disclose the useof streptavidin conjugated with alkaline phosphatase to detecthybridization of biotinylated probes for the human N-myc and B-NGFgenes.

The prior art contains examples of a variety of enzymatic and chemicalmethods for labeling and derivatizing nucleic acid probes. Such probescan be detected by a large number of direct and indirect detectionsystems. This invention relates to methods and compounds for labelingDNA with xanthine and lower alkyl substituted derivatives of xanthineand reagents comprising a large number of different DNA sequencescovalently labeled with xanthine and lower alkyl substituted derivativesof xanthine that are complementary to DNA sequences of a chromosome or aportion of a chromosome. These reagents permit the in situ detection ofthe chromosome. The xanthine- or lower alkyl substituted xanthinederivative-labeled DNA bound to the chromosome or other target canconveniently be detected by immunological techniques.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the preparation oftheophylline-8-N-(5-hydroxypentylamino)-O-succinoyl-O'-(N'-(3-sulfosuccinimidyl))ester.

FIG. 2 illustrates reaction oftheophylline-8-N(5-hydroxypentylamino)-O-succinoyl-O'-(N'-(3-sulfosuccinimidyl))esterwith DNA that has been transaminated with ethylenediamine.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide: 1. methods andcompounds for labeling DNA with xanthine or lower alkyl substitutedderivatives of xanthine, 2. reagents for the in situ detection of achromosome or region of a chromosome, 3. methods for the preparation ofsuch reagents and 4. methods for the use of such reagents for the insitu detection of a chromosome or region of a chromosome.

This invention provides methods and compounds for covalently binding anantigenic label to DNA, as well as the antigen-labeled DNA, wherein theantigenic moiety of the label is xanthine or a lower alkyl substitutedxanthine derivative. By xanthine or a lower alkyl substituted xanthinederivative labels are meant labels of the formula I: ##STR1## whereinR₁, R₂, and R₃ are independently hydrogen or a lower alkyl substituenthaving 1-6 carbon atoms, preferably hydrogen or methyl. Preferred labelsare derivatives of xanthine (wherein R₁, R₂, and R₃ are hydrogen),1-methylxanthine, 3-methylxanthine, 7-methylxanthine,1,3-dimethylxanthine (theophylline), 1,7-dimethylxanthine and1,3,7-trimethylxanthine (caffeine). The most preferred labels arederivatives of theophylline and caffeine.

This invention provides a reagent for in situ detection of nucleic acidsequences comprising one or more DNA sequences complementary to thenucleic acid to be detected wherein the DNA sequences include multiplexanthine or lower alkyl substituted xanthine derivative labelscovalently linked thereto. The preferred labels are theophylline(1,3-dimethylxanthine) and caffeine (1,3,7-trimethylxanthine).

The invention includes a reagent for in situ detection of a chromosomeor region of a chromosome comprising unhybridized DNA sequences havingessentially complementary base sequences with respect to differentportions of the chromosome or chromosome region to be detected andhaving a plurality of cytosine bases that are transaminated, a number ofthe transaminated cytosine bases having a xanthine or lower alkylsubstituted derivative of xanthine label covalently bonded thereto, thenumber of said xanthine- or substituted xanthine derivative-labeledcytosine bases being sufficient for detection by immunologicaltechniques while essentially retaining the specific binding propertiesof the reagent with respect to the chromosome or chromosome region to bedetected.

The invention also includes a method for making a reagent for in situdetection of a chromosome comprising:

(a) disrupting plasmid DNA containing DNA complementary to thechromosome or region of chromosome to be detected into fragments,

(b) transminating the DNA fragments, and

(c) covalently linking xanthine or lower alkyl substituted xanthinederivative labels to the transaminated DNA fragments.

More specifically, the invention includes a method for preparing areagent for in situ detection of a chromosome or region of a chromosomecomprising:

(a) transminating a number of cytosine bases contained in unhybridizedDNA sequences having essentially complementary base sequences withrespect to the chromosome or chromosome region to be detected; and

(b) covalently bonding xanthine or lower alkyl substituted xanthinederivative labels to at least a portion of the transaminated cytosinebases, the portion of cytosine bases having xanthine or lower alkylxanthine substituted derivative labels covalently bonded thereto beingsufficient to generate a detectable xanthine or lower alkyl substitutedxanthine derivative signal while essentially retaining the specificbinding properties of the reagent with respect to the chromosome orchromosome region to be detected.

In addition the invention provides a method for in situ detection of achromosome or region of a chromosome comprising:

(a) adding an excess of blocking DNA to the inventive reagent underhybridizing conditions to bond with nonspecific binding DNA in thereagent, thereby forming a blocked reagent,

(b) contacting the blocked reagent under hybridizing conditions with thechromosome or chromosome region to be detected, and

(c) detecting the binding of the blocked reagent to the chromosome orchromosome region to be detected by immunological techniques.

It is an additional object of the present invention to provide as areagent DNA covalently labeled with xanthine or lower alkyl substitutedderivative of xanthine. It is a specific object of the present inventionto provide a reagent for the in situ detection of a chromosome or regionof a chromosome wherein the reagent contains multiple DNA sequences thatare complementary to different portions of the chromosome or chromosomeregion to be detected. The multiple DNA sequences in the reagent includemultiple xanthine or lower alkyl substituted xanthine derivatives,preferably theophylline or caffeine labels that are covalently linked tothe DNA sequences. These xanthine or lower alkyl substituted derivativeof xanthine, preferably theophylline or caffeine, labels permit the DNAsequences that hybridize with the chromosome or chromosome region ofinterest to be detected using immunological techniques. The xanthine orlower alkyl substituted derivative of xanthine, preferably theophyllineor caffeine labels are covalently linked to any of the bases adenosine,guanosine, cytosine or thymidine comprising the DNA sequences. In apreferred embodiment, the xanthine or lower alkyl substituted derivativeof xanthine, preferably theophylline or caffeine labels are covalentlylinked to a number of transaminated cytosine bases in the DNA sequences.The number of transaminated cytosine bases having xanthine or loweralkyl substituted derivative of xanthine, preferably theophylline orcaffeine labels is sufficient to provide an amount of xanthine or loweralkyl substituted derivative of xanthine, preferably theophylline orcaffeine label detectable by immunological techniques, while at the sametime essentially retaining the specific binding properties of the DNAsequences with respect to the chromosome or chromosome region ofinterest.

It is an additional specific object of the present invention to providemethods for making reagents for the in situ detection of a chromosome orregion of a chromosome. The first step of a preferred method is todisrupt plasmid DNA derived from a phage chromosomal library intofragments. These DNA fragments are transaminated and functional xanthineor lower alkyl substituted derivative of xanthine, preferablytheophylline or caffeine derivatives are then covalently linked to thetransaminated DNA fragments. In the present invention, the number oftransaminated cytosine bases to which xanthine or lower alkylsubstituted derivative of xanthine, preferably theophylline or caffeinelabels are covalently bonded is sufficient to generate a immunologicallydetectable amount of xanthine or lower alkyl substituted derivative ofxanthine, preferably theophylline or caffeine label while at the sametime essentially retaining the specific binding properties of thereagent with respect to the chromosome or chromosome region to bedetected.

It is a further specific object of the present invention to providemethods for the in situ detection of a chromosome or region of achromosome. In general, the preferred methods are carried out bycontacting the reagents of the present invention with the chromosome orchromosome region to be detected under hybridization conditions.

It is an additional object of the present invention to provide methodsfor the detection of any nucleic acid sequence complementary to DNAcovalently labeled with xanthine or lower alkyl substituted derivativeof xanthine. The xanthine or lower alkyl substituted xanthine derivativelabels allow the introduction of a reporter molecule, such as afluorescent or enzyme labeled antibody, to any probe-target complex. Thetarget may be chromosomal DNA, but may additionally be RNA, such asmessenger or ribosomal RNA; extrachromosomal DNA, such as mitochondrialDNA; or prokaryotic DNA, such as bacterial or viral DNA. The DNA probeassay may involve in situ hybridization, solution phase hybridization,hybridization to nucleic acids immobilized on a solid phase, orhybridization to blots of nucleic acid or protein immobilized on any ofa number of transfer media, including but not limited to nitrocellulose,nylon and other synthetic derivatives known in the art.

The presence of the xanthine or lower alkyl substituted derivative ofxanthine label such as theophylline or caffeine is detected byimmunological techniques. Xanthine or lower alkyl substitutedderivatives of xanthine are haptens to which antibodies can be raised.For example, an anti-theophylline antibody is reacted with thetheophylline moiety of the reagent bound to the target chromosome. Asecond antibody specifically reactive to the anti-theophylline antibodyand which is labeled with an enzyme or fluorescent marker is then boundto the anti-theophylline antibody to indirectly determine the binding ofthe theophylline containing DNA to the chromosome. Those skilled in theimmunological arts recognize a large number of techniques forimmunologically determining the presence of a hapten such astheophylline or caffeine.

In a preferred embodiment, an excess of blocking DNA is added to thereagent under hybridizing conditions. This blocking DNA then binds withany nonspecific binding DNA in the reagent, thereby forming a blockedreagent. This blocked reagent can then be contacted with the chromosomeor chromosome region of interest under hybridizing conditions.

The present invention additionally includes a reagent comprising DNAhaving one or more xanthine or lower alkyl substituted xanthine groupscovalently bound thereto wherein the xanthine or lower alkyl substitutedxanthine derivative label is detectable by immunological techniques andthe DNA retains its binding properties.

The present invention also includes a compound which istheophylline-8-N-(5-hydroxypentylamino)-O-succinoyl-O'-(N'-(3-sulfosuccinimidyl))ester.

The present invention also includes a compound which iscaffeine-8-N-(5-hydroxypentylamino)-O-succinoyl-O'-(N'-(3-sulfosuccinimidyl))ester.

The present invention also includes a reagent for in situ detection ofnucleic acid sequences comprising one or more DNA sequencescomplementary to the nucleic acid to be detected wherein the DNAsequences include multiple theophylline or caffeine labels covalentlylinked thereto.

Further objects and preferred embodiments of the present invention willbe discussed in the following description of the preferred embodimentsand claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention encompasses methods and compounds for labeling DNA withxanthine and lower alkyl substituted derivatives of xanthine, andreagents and methods for the in situ identification of chromosomes orregions of chromosomes. The invention encompasses a reagent comprising amultiplicity of DNA sequences complementary to different portions of thechromosome or chromosome regions to be detected wherein the DNAsequences are aminated at multiple sites and labeled with xanthine orlower alkyl substituted derivatives of xanthine at the aminated sites bycovalent linkage of a functionalized derivative of xanthine or a loweralkyl substituted derivative of xanthine.

The sources of the DNA sequence used in the invention include but arenot limited to DNA isolated from specific chromosomes, or libraries ofsuch DNA, prepared by methods well known to those with skill in the art.The individual chromosomes from which DNA is isolated can be prepared byany of a number of standard methods, such as flow cytometry of microcellor somatic cell hybrids, or by direct isolation from individualmetaphase or interphase cells. Another source of such DNAs are librariesof specific chromosomal DNA, prepared by standard methods and availablefrom traditional sources known to those in the art, such as the AmericanType Culture Collection (ATCC) or other repositories of human or othercloned genetic material. While a large number of chromosome librariesare available from the ATCC, representative libraries are:

    ______________________________________    Human    Chromosome    Library        ATCC No.    ______________________________________     1             57738     1             57753     1             57754     2             57716     2             57744     3             57717     3             57748     3             57751     4             57719     4             57718     4             57700     4             57745     5             57720     5             57746     6             57721     6             57701     7             57722     7             57755     8             57723     8             57702     9             57724     9             57705    10             57725    10             57736    11             57726    11             57704    12             57727    12             57736    13             57728    13             57705    14             57739    14             57706    14/15          57707    15             57729    15             57740    15             57737    16             57765    16             57730    16             57749    16             57758    17             57741    17             57759    18             57742    18             57710    19             57731    19             57766    19             57711    20             57732    20             57712    21             57743    21             57713    22             57733    22             57714    X              57750    X              57734    X              57752    X              57747    Y              57735    Y              57715    ______________________________________

The ATCC deposits are available from the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. The inventioncontemplates that such DNA sequences may also be synthesized in vitro byany of a number of enzymatic means known to those in the art. Also seean article entitled Human Chromosome-Specific DNA Libraries,Biotechnology 4: 537 (1986), which describes the preparation of humanchromosome libraries.

DNA used in the invention is isolated from these sources by methodswhich are well known to those skilled in the art. This DNA is thenreduced to a heterogenous mixture of variably sized fragments by any ofa number of physical, chemical or enzymatic treatments, including butnot limited to sonication, limited DNase I digestion, limited mung beannuclease digestion, and shearing of DNA through a narrow-gauge needle.The resulting mixture of DNA fragments will be within a size range of100-500 basepairs (bps) in length, although the preferred length of theaverage size of a fragment is about 300 bps. These procedures provide alarge number of DNA sequences complementary to different portions of thechromosomes to be detected. In fact thousands if not tens of thousandsof DNA sequences complementary to different portions of the chromosomeDNA are provided.

The DNA fragments are derivatized by any of a number of chemical meansknown to those in the art, preferably by transamination of the carbon 4(C-4) atom amino group of the nucleotide base cytosine. Thederivatization will result in the addition of a variety of diaminecompounds at the C-4 position in this base, including but not limited tosuch compounds as hydrazine and alkylene diamines having 2 to 10 carbonatoms such as ethylenediamine, as well as compounds including but notlimited to amino acids, peptides, ether derivatives, or any of a numberof other organic or inorganic linker molecules. The DNA fragments have5-25% of the cytosine residues contained therein transaminated, with anoptimum transamination of 12-24%. About 1-7% of the total nucleotideresidues are transaminated, with an optimum of 2-5% of the total basestransaminated.

The transaminated DNA sequences are covalently linked to any of a numberof xanthine or lower alkyl substituted xanthine derivative compoundsthat have a reactive functional group capable of covalent bond formationwith the transaminated DNA sequence. Preferred compounds that can beused to respectively theophylline or caffeine label the transaminatedDNA sequences aretheophylline-8-N-(5-hydroxypentylamino)-O-succinoyl-O'-(N'-(3sulfosuccinimidyl))esterorcaffeine-8-N-(5-hydroxypentylamino)-O-succinoyl-O'-(N'-(3-sulfosuccinimidyl))ester.The transaminated DNA sequences are reacted with a 100-300-fold molarexcess of functionalized theophylline or caffeine compound, preferablyabout a 150-fold molar excess. About 80-100% of transaminated sites aretheophylline- or caffeine-labeled.

Xanthine or lower alkyl substituted xanthine derivative labeled DNAsequences are used for in situ hybridization to target chromosomes. Forthe purposes of this invention "in situ" means that the chromosomes areexposed from the cell nucleus without substantial disruption orrelocation of the chromosomes with respect to each other, and with thechromosomes being accessible to the xanthine or lower alkyl substitutedderivative of xanthine, preferably theophylline or caffeine, labeled DNAprobes. Targets for this hybridization include but are not limited tochromosomes or regions of chromosomes in normal, diseased or malignanthuman or other animal or plant cells, either interphase or at any stageof meiosis or mitosis, and either extracted or derived from living orpostmortem tissues, organs or fluids; germinal cells including sperm andegg cells, seeds, pollen, or zygotes, embryos, chronic or amnioticcells, or cells from any other germinating body; cells grown in vitro,from either long-term or short-term culture, and either normal,immortalized or transformed; inter- or intraspecific hybrids ofdifferent types of cells or differentiation states of these cells;individual chromosomes or portions of chromosomes, or translocated,deleted or other damaged chromosomes, isolated by any of a number ofmeans known to those with skill in the art, including libraries of suchchromosomes cloned and propagated in prokaryotic or other cloningvectors, or amplified in vitro by means well known to those with skill;or any forensic material, including but not limited to semen, blood,hair or other samples.

Prior to hybridization, the labeled DNA sequences are reacted with anexcess of corresponding unlabeled DNA or reassociated fraction ofunlabeled DNA to bind with nonspecific binding DNA in the sample,thereby blocking the nonspecific DNA to provide a blocked reagent. Thisblocking DNA is used at a concentration of 1-10 micrograms per 10microliters of total genomic DNA, with a preferred range depending onthe hybridized chromosome of between 2.25 and 6.75 micrograms per 10microliters, or between 1 and 4 micrograms per 10 microliters,preferably about 1.3 micrograms per 10 microliters. The blocking DNA maybe human placental DNA or Cotl DNA (Cotl DNA supplied by LifeTechnologies, Gaithersburg, Md., Cat. #5279SA) is prepared bymechanically shearing total human genomic DNA to an average size of lessthan 400 base pairs. This material is denatured and then rehybridizedfor a period sufficient to render a large fraction of the highlyrepeated DNA sequences doublestrained. The mixture of double andsingle-stranded DNA species are treated with nuclease S1, a nucleasethat specifically degrades single-stranded DNA to mono- andoligo-nucleotides. Undigested, double stranded Cotl DNA is recoveredfrom this mixture. Chromosomes are preferably stained so as to bevisible with any of a number of counterstains, including but not limitedto propidium iodide, quinacrine, ordistamycin/4,6-diamino-2-phenylindole (DAPI) or with a non-fluorescentdye such as Giemsa. These counterstains permit visualization of all ofthe chromosomes.

Thus this invention provides a mixture of a large number of xanthine orlower alkyl substituted derivatives of xanthine labeled DNA sequences,preferably theophylline or caffeine labels that are complementary todifferent portions of the chromosomes to be detected. The methods ofthis invention provide thousands, if not tens of thousands of differentDNA sequences that are complementary to the chromosome being detectedand are labeled with xanthine or lower alkyl substituted derivatives ofxanthine, most preferably theophylline or caffeine. Hybridization ofthis large number of different labeled DNA sequences to a substantialportion of the chromosomes permits detection of the chromosome byreacting the hybridized, labeled DNA with an antibody against the labeland in turn binding the antibody bound to the label with an antibodylabeled with an enzyme or fluorescent marker and which is specific forthe antibody bound to the label. Those skilled in the immunological artswill recognize a large number of immunological techniques by which thexanthine or lower alkyl substituted xanthine labels such as theophyllineor caffeine can be detected. Xanthine or lower alkyl substitutedderivatives of xanthine such as theophylline and caffeine, for example,are haptens to which antibodies are raised by well known techniques.Techniques for labeling antibodies with fluorescent markers such asfluorescein, rhodamine and the like are well known. It is also wellknown to label antibodies with enzymes such as horseradish peroxidaseand alkaline phosphatase. For example, the xanthine or lower alkylsubstituted derivatives of xanthine label, such as theophylline label,on the DNA fragments hybridized to the target chromosome could bedetected by reaction directly with a fluorescent or enzyme labeledanti-theophylline antibody. The complex is represented as follows:

Chromosome DNA/complementary DNA--theophylline labeled/anti-theophyllineantibody (fluorescent or enzyme label)

In another configuration, the theophylline label is reacted with ananti-theophylline antibody, which in turn is bound to an antibody to theanti-theophylline antibody that has a fluorescent or enzyme label. Forexample, rabbit anti-theophylline antibody is first bound to thetheophylline label and is followed by goat antirabbit antibody that hasa fluorescent or enzyme label. This configuration is represented asfollows:

Chromosome DNA/complementary DNA--theophylline labeled/rabbitanti-theophylline antibody/goat anti-rabbit antibody with a fluorescentor enzyme label.

The fluorescent or enzyme labels are detected by standard techniques.Those skilled in the immunological arts will recognize a large number ofassay configurations, fluorescent labels, and enzyme labels fordetecting haptens such as xanthine or lower alkyl substituted xanthinederivatives, particularly theophylline or caffeine labels. Advantage mayalso be taken of other specific binding substances such as avidin/biotinin configuring assays for the xanthine or lower alkyl substitutedxanthine derivatives, particularly theophylline or caffeine label.

The present invention is further illustrated by the following examples.However, many embodiments not specifically described herein neverthelessfall within the spirit and scope of the present invention.

EXAMPLE 1

Isolation of Human Chromosome--Specific DNA Probes

Human chromosome-specific DNA probes were obtained as recombinant phagelibraries from Lawrence Livermore National Laboratories (LLNL)constructed as described in Van Della, M. A. et al. (Biotechnology 4:537-552, 1986). These libraries were amplified by growth on an E. colihost strain. The amplified phage were purified, their DNA was extracted,and this DNA was digested with the restriction enzyme Hind III. InsertDNA was purified away from the lambda vector DNA and cloned into theHind III site of the plasmid vector pBS (Strategene, La Jolla, Calif.).The resulting plasmids were transformed into an E. coli strain, DH5α(Bethesda Research Libraries, Gaithersburg, Maryland).

The plasmid libraries exemplified herein are ATCC #'s 57738, 57753 and57754 (Chromosome 1); ATCC numbers 57749, 57748, and 57751 (Chromosome3); ATCC numbers 57723 and 57702 (Chromosome 8) and ATCC numbers 57727and 57736 (Chromosome 12). The libraries are stored as 1 ml aliquots offrozen cells. These vials have been used as the primary source for theproduction of seed stocks for fermentation.

Bacteria were grown by fermentation. The seed stock obtained from ATCCwas cultured at 37° C. for 24 hr. on 1.6% agar plates containingampicillin (200 microgram/ml) and YT broth, which contains 8 grams perliter (g/l) of Bacto Tryprone (Difco), 5 g/1 of Bacto Yeast Extract(Difco), 15 g/l of Bacto Agar (Difco), and 5 g/l of sodium chloride. Thecultured cells were harvested with 4 ml containing 16 g/l of BactoTryprone (Difco), 10 g/l of Bacto Yeast Extract (Difco) and 5 g/l ofsodium chloride, and 4 ml of 20% glycerol was added to each harvest. TheE. coil cell culture was quickly frozen in 0.5 ml aliquots by submergingthe vials in liquid nitrogen and stored at -80° C. until use.

The fermenter inoculum was prepared in 350 ml by culturing the seedculture in a Casamino Acid medium which contains 13.2 g/l Na₂ HPO4-7H₂O, 3.0 g/l KH₂ PO₄, 0.05 g/l NaCl, 1.0 g/l NH₄ Cl, 10.0 g/l CasaminoAcids (Difco); 0.03 g/l MgSO₄, 0.004 g/l CaCl₂ -2H₂ O, 3.0 g/l glucose,0.025 g/l Thiamine-HCl, 0.0054 g/l FeCl₃, 0.0004 g/l ZnSO₄, 0.0007 g/lCoCl₂, 0.0007 g/l Na₂ MoO₄, 0.0008 g/l CuSO₄, 0.0002 g/l H₂ BO₃, and0.0005 g/l MnSO₄ in a 2 liter baffled shaker flash at pH 7 and 37° C.The 350 ml culture was used to inoculate 4.2 liters of fermentationmedia containing 1% glucose, 13.2 g/l Na₂ HPO4-7H₂ O, 3.0 g/l KH₂ PO₄,0.05 g/l NaCl, 1.0 g/l NH₄ Cl, 10.0 g/l Casamino Acids (Difco), 0.03 g/lMgSO₄, 0.004 g/l CaCl₂ -2H₂ O, 0.025 g/l Thiamine-HCl, 0.0054 g/l FeCl₃,0.0004 g/l ZnSO₄, 0.0007 g/l CoCl₂, 0.0007 g/l Na₂ MoO₄, 0.0008 g/lCuSO₄, 0.0002 g/l H₂ BO₃, and 0.0005 g/l MnSO₄.

Bacterial cells were harvested employing a membrane cell-concentratorand a high speed centrifuge immediately after completion of thefermentation. The fermented cell broth was concentrated from 5 liter toapproximately 800 ml employing a 0.45 micron (μm) membrane filter (2square feet). The cell concentrate was then centrifuged at 7,000×g for10 minutes in a refrigerated centrifuge. The bacterial cell pellets arerecovered after discarding the supernatant.

Plasmid DNA was extracted from bacterial cell pellets. The cells werethoroughly resuspended in 3 times the cell pellet mass (M) (inmilliliters) of a solution containing 50 mM glucose (filter sterilized),10 mM NaEDTA (pH 7.5-8.0), and 25 mM Tris-HCl (pH 8.0). The cells werelysed with vigorous swirling after the addition of 6×M (in milliliters)in a solution containing 0.2M NaOH, and 1% (w/v) sodium dodecylsulfate(SDS). When the solution cleared, 4.5×M (in milliliters) of a solutioncontaining 55.5 ml of glacial acetic acid and 147.5 grams of potassiumacetate in a final volume of 500 ml was mixed thoroughly resulting inthe production of a flocculent precipitate. The supernatant wasseparated from the flocculent precipitate and this supernatantcentrifuged for 15 minutes at 7000×g to remove residual precipitate.

Nucleic acid was precipitated from the supernatant with one volume ofethanol followed by centrifugation for 10 minutes at 7000×g, and thenucleic acid pellets were resuspended in a total of 0.54×M (inmilliliters). The nucleic acid was then extracted with 1/2 volume ofneutralized phenol and 1/2 volume of chloroform and precipitated withtwo volumes of ethanol. The nucleic acid was resuspended in 0.3×M (inmilliliters) of a solution of 50 mM Tris HCl (pH 7.0) and 100 mM sodiumacetate. 0.77724 (in microliters) of 10 mg/ml RNase (heat treated) wasthen added and allowed to digest for 30 minutes at room temperature orovernight at 4° C. 0.615×M (in microliters) of a solution of ProteinaseK (20 mg/ml) was then added and incubated at 55° C. for three hours. DNAwas extracted with 1/2 volume of neutralized phenol and 1/2 volume ofchloroform and precipitated with two volumes of ethanol.

DNA was resuspended in 0.415×M (in milliliters) of water, and 0.05×Mmilliliters of 5M NaCl and 0.155×M milliliters of 50% (w/v)polyethyleneglycol (PEG) (molecular weight 6000-8000) were added,incubated on ice water for one hour and precipitated by centrifugationfor 15 minutes at 7,000×g. The DNA was resuspended in 0.04×M millilitersof water and 1/10 volume of 3M sodium acetate and extracted with 1/2volume of neutralized phenol and 1/2 volume of chloroform andprecipitated with two volumes of ethanol. The purified DNA wasresuspended in 0.0476×M milliliters of deionized H₂ O. The DNAconcentration was determined by fluorometry.

Finally, the purified DNA was disrupted into small fragments ofapproximately 300 base pairs by sonication using a Branson Sonifier 450(Danbury, Conn.). This size of fragments has been empirically determinedto be the optimum for DNA probes used for in situ hybridization. Fourmilligrams of the purified plasmid DNA prepared above was resuspended in2 mls of water and immersed in a dry ice/ethanol bath to prevent boilingduring sonication. The microtip of the sonication device was immersed inthis solution until the tip was 2-5 mm from the bottom of the tube.Sonication was carried out at an output power of 25-30 watts,discontinuously, with an 80% duty cycle (on 80% of time, off 20% oftime), for a period of 5 minutes. Following sonication, the DNA wasprecipitated by the addition of 0.2 ml of 3M sodium acetate (pH 5.5) and4 ml of ethanol. The precipitate was recovered by centrifugation for 5minutes at 8,000×g and vacuum dried.

EXAMPLE 2

Bisulfite Catalyzed Transamination of DNA

DNA was transaminated by the addition of ethylenediamine to the C4carbon atom of the base cytosine. This reaction is catalyzed by sodiumbitsulfite. Approximately 8 to 24% of the available deoxycytidinenucleotide sites are aminated for labeling with xanthine or lower alkylsubstituted xanthine derivatives, particularly theophylline or caffeine.

To prepare the bisulfite buffer, 1.7 ml of fuming HCl was slowly addedto 1 ml deionized H₂ O on ice. 1 ml fresh ethylenediamine (Sigma cat.#E-4379) was then slowly added on ice. After dissolution of theethylenediamine, the solution was warmed to room temperature and 0.475 gsodium metabisulfite (Aldrich Cat. #25,555-6) was added. Fuming HCl wasthen slowly added to the bisulfite mixture until the pH reached 7.0.Deionized water was added to a final volume of 5.0 ml.

To transaminate DNA, 1 milligram of sonicated DNA was resuspended in 0.3ml H₂ O. The DNA was denatured by boiling at 100° C. for 5 minutes thenquickly chilled in an ice water bath. The transamination reaction wasinitiated by the addition of 0.3 ml of this DNA solution to 2.7 ml ofbisulfite buffer, and the reaction was incubated at 37° C. for 2 days.The DNA solution was desalted by routine dialysis against 5-10 mM sodiumborate (pH 8.0). After dialysis, 0.3 ml of 3M sodium acetate (pH 5.5)was added to the dialysate. The aminated DNA was precipitated with 2.5volumes of ethanol and recovered after centrifugation at 8,000×g for 10minutes. The pellets were vacuum dried and rehydrated at a concentrationof 3 mg/ml DNA. This solution was stored at -80° C. until use.

EXAMPLE 3

Preparation of theophylline-8-N-(5-hydroxypentylamino)-O-succinoylO'-(N'-(3-sulfosuccinimidyl)) ester

A solution of 5.16 grams of 8-bromotheophylline and 5.15 grams of5-amino-1 pentanol in 18 ml of p-xylene was refluxed for 18 hours. Thereaction solution was allowed to cool and upon cooling to roomtemperature formed a solid/liquid mixture. The xylene was decanted andthe solid was washed with pentane (3×40 ml). The resulting solid wasstirred in 20 ml of water for 30 minutes and filtered to collect thesolid material. The collected solid material was washed with water (3×20ml), and dried to provide 3.73 g of8-(5-hydroxypentylamino)theophylline. To a solution of 1.12 g of8-(5-hydroxypentylamino)theophylline in 15 ml of anhydrousN,N-dimethylformamide (DMF) was added 0.516 g of succinic anhydride and0.2 g of 4-dimethylamino pyridine (DMAP). The mixture was stirred with amagnetic stirrer overnight at room temperature under anhydrousconditions. The colorless solid that precipitated was collected byvacuum filtration, washed with dichloromethane and air dried to provide1.20 g of theophylline-8-N-(5-hydroxypentylamino)-O-succinic acid ester.This solid was recrystallized from a boiling 1:1 propanol/water mixtureand dried over calcium sulfate to provide a colorless solid.

To a solution of 0.725 g oftheophylline-8-N-(5-hydroxypentylamino)-O-succinic acid ester in 15 mlof anhydrous dimethylformamide was added 0.414 g of3-sulfo-N-hydroxysuccinimide (Sulfo-NHS).

A solution of 0.488 g of dicyclohexyl-carbodiimide (DCC) in 2 ml ofanhydrous DMF was added to the above solution. The resulting mixture wasstirred with a magnetic stirrer at room temperature for 16 hours. Thereaction mixture was cooled in an ice bath for 1 hour, then filteredunder vacuum to remove dicyclohexylurea. The filtrate was evaporated invacuo (2 torr, 30° C.) to provide a viscous colorless oil. This oil wastreated with 40 ml of anhydrous ethanol and a solid materialprecipitated. The precipitated material was collected by filtration anddried over anhydrous calcium sulfate to provide 0.450 g oftheophylline-8-N-(5-hydroxypentylamino)-O-succinoyl-O'-(N'-(3-sulfosuccinimidyl))ester,(NHS-theophylline). This reaction scheme is illustrated in FIG. 1.Labeling of aminated DNA with NHS-theophylline is shown in FIG. 2.

EXAMPLE 4

Preparation of 8-(5-hydroxypentylamino) caffeine,caffeine-8-N(5-hydroypentylamino)-O-succinic acid ester and caffeine-8-N(5-hydroxypentylamino)-O-succinoyl-O'-(N'-3-sulfosuccinimidyl))ester,(NHS-caffeine)

Beginning with 8-(5-hydroxypentylamino) theophylline in Example 3, 1.0ml of dimethylsulfate (DMS) was added to 1.40 g of8-(5-hydroxypentylamino) theophylline in 20 ml of water containing 0.3 gof sodiumhydroxide. The 1.0 ml DMS was added in 0.1 ml increments overof hour at: room temperature. A colorless crystalline solid separatedand was collected by filtration. This solid was dried to provide 0.850 gof 8-(5-hydroxypentylamino) caffeine.

Following the procedures in Example 3 and using equivalent quantities,the 8-(5-hydroxypentylamino) caffeine is converted tocaffeine-8-N(5-hydroypentylamino)-O-succinic acid ester andcaffeine-8-N-(5-hydroypentylamino)-O-succinoyl-O-(N'-(3-sulfosuccinimidyl))ester,(NHS-caffeine).

EXAMPLE 5

DNA Labeling--Theophylline

Chromosome-specific DNA probes to human chromosome 8 of average lengthof about 300 bp obtained by the procedure of Example 1 were derivatizedwith the bisulfite catalyzed transamination with ethylenediamine asdescribed in Example 2. Approximately 5% of the bases were aminated. Asolution of aminated DNA (100 μg total DNA) in a plastic 1.5 mlcentrifuge tube was evaporated under reduced pressure. To this solutionwas added 0.5 ml of 0.2M 3- N-morpholino!propane sulfonic acid (MOPS)buffer and then 100 microliters of NHS-theophylline (26mg/mlN,N-dimethylformamide) was added to the residue and the mixture wasincubated overnight at 25° C. 50 μl of 3M sodium acetate (pH 5.5) wereadded followed by 1.5 ml of ice cold ethanol and the mixture wasincubated for at least 2 hours at -20° C. The solution was subjected tocentrifugation for 10 minutes at 10,000×g. The pellet was washed twicewith 0.6 ml of ice cold ethanol and then dissolved in 100 μl of sterilewater. Two Sephadex G-50 Select D chromatography columns (5 Prime→3Prime, Inc.) with a bed volume of 0.8 ml were prepared. 50 μl of thedissolved pellet was applied to each column and centrifuged for 4minutes at 10,000×g. 10 μl of the purified DNA was diluted with 490 μlof 20 mM NaOH and the optical density was determined at 260 nm to assessDNA concentration. The purified DNA was diluted with water to provide aworking concentration of DNA of 100 micrograms per ml.

In situ Hybridization Procedure.

The theophylline labeled DNA probe was tested by in situ hybridization.The target DNA consisted of cultured normal white blood cells that werearrested in metaphase. The cells were dropped onto a glass microscopeslide from a distance of about three feet to break open the nuclei.Before hybridization, the slide was placed in a denaturing solution of70% formamide/0.3M NaCl/30 mM sodium citrate (pH 7) at 70° C. The slideas then dehydrated by passage through solutions of 70%, 85% and 100%ethanol (2 min each).

The hybridization mixture that was placed on each slide consisted of 50%formamide/0.3M NaCl/30 mM sodium citrate (pH 7). Human placental DNA(2.25 micrograms/10 microliters) was used as blocking DNA. Thetheophylline labeled DNA was added at a concentration of 10 ng/μl. Tenmicroliters of the complete hybridization mixture was denatured byheating at 70° C. for 10 minutes. The probe and blocking DNA wasprehybridized for 60 minutes at 37° C. The mixture as then applied tothe slide, covered with a coverslip and sealed with rubber cement.Hybridization proceeded overnight at 37° C. in a humidified chamber.

The next day, the excess probe was removed by washing the slide threetimes for 15 minutes at 45° C. in 50% formamide/0.3M NaCl/30 mM sodiumcitrate (pH 7), then in 0.3M NaCl/30 mM sodium citrate (pH 7) and in0.1M sodium phosphate (pH 7)/0.1% NP40 detergent(octylphenoxypolyethoxyethanol, which is a nonionic surfactant sold byCalbiochem, La Jolla, Calif.) (PN buffer) each for 15 minutes at 45° C.The slide was washed 2 times in PN buffer at 25° C. 2 minutes each wash.The slide was blocked for 1 hour in 0.1M sodium phosphate (pH 7)/0.1%NP40 detergent/5% nonfat dry milk (PNM buffer). The slide was incubated1 hour at 25° C. in rabbit anti-theophylline (Sigma) diluted 1:250 inPNM buffer. The slide was washed in PN buffer, 3 times for 5 minuteseach at 25° C. The slide was then incubated for 1 hour in alkalinephosphatase-anti-rabbit immunoglobulin G (Sigma) diluted 1:250 in PNMbuffer. The slide was washed in PN buffer 3 times for 5 minutes each at25° C. The slide was then incubated in 17 ml of BCIP/NBT a solutioncontaining 4 μl/ml of nitro blue tetrazolium (NBT) and 3 mg/ml of5-bromo-4-chloro-3-indolyl-phosphate (BCIP, as provided by Chemprobe)!for 30 minutes at 37° C. The slide was rinsed in distilled water, airdried, and viewed with a microscope.

In situ Hybridization Results

The alkaline phosphatase label catalyses the conversion of the BCIP/NBTsubstrate into a dark blue dye which appears black when viewed with themicroscope. Nontarget chromosomes either appear colorless or can becounterstained blue with a Giemsa stain preparation.

Following substantially the above procedures, results for Chromosomes 1,3, and 12 were also obtained.

    ______________________________________                    Visual Description    Chromosome Number Specificity                                Intensity    ______________________________________    1                 +++       +++    3                 +++       ++    8                 +++       +++    12                ++        ++    ______________________________________     Code:     Specificity: (-) none apparent, (+) slightly heavier staining in target     chromosomes, (++) reasonable specificity, (+++) good specificity.     Intensity: (-) not visible, (+) visible under phase only, (++) dark     staining, visible under transmission, (+++) very dark staining, refractil     under phase.

EXAMPLE 6

DNA Labeling--Caffeine

Chromosome-specific DNA probes to human chromosome 4 of average lengthof about 300 bp obtained by the procedure of Example 1 were derivatizedwith the bisulfite catalyzed transamination with ethylenediamine asdescribed in Example 2. Approximately 4.3% of the bases were aminated. Asolution of aminated DNA (100 μg total DNA) in a plastic 1.5 mlcentrifuge tube was evaporated under reduced pressure. 0.5 ml of 3-N-morpholino!propane sulfonic acid (MOPS) buffer, then 100 μl ofNHS-caffeine (3 mg/ml N,N-dimethylformamide) was added to the residueand the mixture was incubated overnight at 25° C. 50 μl of 3M sodiumacetate (pH 5.5) were added followed by 1.5 ml of ice cold ethanol andthe mixture was incubated for at least 2 hours at -20° C. The solutionwas subjected to centrifugation for 10 minutes at 10,000×g. The pelletwas washed twice with 0.6 ml of ice cold ethanol and then dissolved in100 μl of sterile water. Two Sephadex G-50 Select D chromatographycolumns (5 Prime→3 Prime, Inc.) with a bed volume of 0.8 ml wereprepared. 50 μl of the dissolved pellet was applied to each column andcentrifuged for 4 minutes at 10,000×g. 10 μl of the purified DNA wasdiluted with 490 μl of 20 mM NaOH and the optical density was determinedat 260 nm to assess DNA concentration. The purified DNA was diluted withwater to provide a working concentration of DNA of 100 micrograms perml.

In situ Hybridization Procedure

The caffeine labeled DNA probe was tested by in situ hybridization. Thetarget DNA consisted of cultured normal white blood cells that werearrested in metaphase. The cells were dropped onto a glass microscopeslide from a distance of about three feet to break open the nuclei.Before hybridization, the slide was placed in a denaturing solution of70% formamide/0.3M NaCl/30 mM sodium titrate (pH 7) at 70° C. The slideas then dehydrated by passage through solutions of 70%, 85% and 100%ethanol (2 min each).

The hybridization mixture that was placed on each slide consisted of 50%formamide/0.3M NaCl/30 mM sodium citrate (pH 7). Human placental DNA(2.25 micrograms/10 microliters) was used as blocking DNA. The caffeinelabeled DNA was added at a concentration of 10 ng/μl. Ten microliters ofthe complete hybridization mixture was denatured by heating at 70° C.for 10 minutes. The probe and blocking DNA mixture was prehybridized for60 minutes at 37° C. The mixture as then applied to the slide, coveredwith a coverslip and sealed with rubber cement. Hybridization proceededovernight at 37° C. in a humidified chamber.

The next day, the excess probe was removed by washing the slide threetimes for 15 minutes at 45° C. in 50% formamide/0.3M NaCl/30 mM sodiumcitrate (pH 7), then in 0.3M NaCl/30 mM sodium citrate (pH 7) and in0.1M sodium phosphate (pH 7)/0.1% NP40 detergent(octylphenoxypolyethoxyethanol, which is a nonionic surfactant sold byCalbiochem, La Jolla, Calif.) (PN buffer) each for 15 minutes at 45° C.The slide was washed 2 times in PN buffer at 25° C., 2 minutes eachwash. The slide was incubated 20 minutes at 25° C. in rabbitanti-caffeine (Western Chemical Research Corporation, Ft. Collins,Colo.) diluted 1:250 in PNM buffer. The slide was washed in PN buffer, 3times for 2 minutes each at 25° C. The slide was then incubated for 20minutes in horse radish peroxidose-anti-rabbit immunoglobulin G (Sigma)diluted 1:250 in PNM buffer. The slide was washed in PN buffer 3 timesfor 2 minutes each at 25° C. The slide was then incubated intetramethylbenzidine (TMB) for 30 minutes at 37° C. The slide was rinsedin distilled water, air dried, and viewed with a microscope.

In situ Hybridization Results

The horse radish peroxidase label catalyses the conversion of the TMBsubstrate into a brilliant blue dye which appears blue-green when viewedwith the microscope. Nontarget chromosomes either appear colorless orcan be counterstained red with a Safranin 0 (Sigma, 0.5% aqueous)preparation.

    ______________________________________                    Visual Description    Chromosome Number Specificity                                Intensity    ______________________________________    4                 +++       ++    ______________________________________     Code:     Specificity: (-) none apparent, (+) slightly heavier staining in target     chromosomes, (++) reasonable specificity, (+++) good specificity.     Intensity: (-) not visible, (+) visible under phase only, (++) dark     staining, visible under transmission, (+++) very dark staining, refractil     under phase.

EXAMPLE 7

DNA Labeling--Theophylline on Centromeric Probe

Chromosome-specific DNA probes to human chromosome 8 of average lengthof about 300 bp obtained by the procedure of Example 1 were derivatizedwith the bisulfite catalyzed transamination with ethylenediamine asdescribed in Example 2. Approximately 3.5% of the bases were aminated. Asolution of aminated DNA (100 μg total DNA) in a plastic 1.5 mlcentrifuge tube was evaporated under reduced pressure. 0.5 ml of 0.2M 3-N-morpholino! propane sulfonic acid (MOPS) buffer and then 100 μl ofNHS-theophylline (30 mg/ml N,N-dimethylformamide) was added to theresidue and the mixture was incubated overnight at 25° C. 50 μl of 3Msodium acetate (pH 5.5) were added followed by 1.5 ml of ice coldethanol and the mixture was incubated for at least 2 hours at -20° C.The solution was subjected to centrifugation for 10 minutes at 10,000×g.The pellet was washed twice with 0.6 ml of ice cold ethanol and thendissolved in 100 μl of sterile water. Two Sephadex G-50 Select Dchromatography columns (5 Prime→3 Prime, Inc.) with a bed volume of 0.8ml were prepared. 50 μl of the dissolved pellet was applied to eachcolumn and centrifuged for 4 minutes at 10,000×g. 10 μl of the purifiedDNA was diluted with 490 μl of 20 mM NaOH and the optical density wasdetermined at 260 nm to assess DNA concentration. The purified DNA wasdiluted with water to provide a working concentration of DNA of 100micrograms per ml.

In situ Hybridization Procedure

The theophylline labeled DNA probe was tested by in situ hybridization.The target DNA consisted of cultured normal white blood cells that werearrested in metaphase. The cells were dropped onto a glass microscopeslide from a distance of about three feet to break open the nuclei.Before hybridization, the slide was placed in a denaturing solution of70% formamide/0.3M NaCl/30 mM sodium citrate (pH 7) at 70° C. The slideas then dehydrated by passage through solutions of 70%, 85% and 100%ethanol (2 min each).

The hybridization mixture that was placed on each slide consisted of 50%formamide/0.3M NaCl/30 mM sodium citrate (pH 7). Human placental DNA(2.25 μg/10 μl) was used as blocking DNA. The theophylline labeled DNAwas added at a concentration of 30 ng/μl. Ten microliters of thecomplete hybridization mixture was denatured by heating at 70° C. for 10minutes. The mixture as then applied to the slide, covered with acoverslip and sealed with rubber cement. Hybridization proceededovernight at 37° C. in a humidified chamber.

The next day, the excess probe was removed by washing the slide threetimes for 15 minutes at 40° C. in 50% formamide/0.3M NaCl/30 mM sodiumcitrate (pH 7), then in 0.3M NaCl/30 mM sodium citrate (pH 7) and in0.1M sodium phosphate (pH 7)/0.1% NP40 detergent(octylphenoxypolyethoxyethanol, which is a nonionic surfactant sold byCalbiochem, La Jolla, Calif.) (PN buffer) each for 15 minutes at 45° C.The slide was washed 2 times in PN buffer at 25° C., 2 minutes eachwash. The slide was incubated 20 minutes at 25° C. in rabbitanti-theophylline (Sigma) diluted 1: 250 in PNM buffer. The slide waswashed in PN buffer, 3 times for 2 minutes each at 25° C. The slide wasthen incubated for 20 minutes in horse radish peroxidase-anti-rabbitimmunoglobulin G (Sigma) diluted 1:250 in PNM buffer. The slide waswashed in PN buffer 3 times for 2 minutes each at 25° C. The slide wasthen incubated in tetramethylbenzidine (TMB, Vector Laboratories) for 5minutes according to the manufacturer's instructions. The slide wasrinsed in distilled water, air dried, and counterstained in aqueoussafranin (0.5% w/v) for 5 minutes at 25° C.

Quantitative In situ Hybridization Results

The horse radish peroxidase label catalyses the conversion of the TMBsubstrate into a brilliant blue dye. Metaphase chromosomes labeled withthe chromosome 8 centromeric probe appear as one pair of chromosomeswhich are stained darkly at the center. In interphase nuclei, chromosome8 centromeric staining appears as discrete spots, dark blue against ared background. It is also possible to count the dots per nucleus. As ademonstration of the accuracy and specificity of this method, 100 nucleiwere counted and the distribution frequency for spot numbers was foundas follows:

    ______________________________________    Number of Spots                   % Nuclei Containing    ______________________________________    0              1    1              6    2              87    3              6    ______________________________________

We claim as our invention:
 1. A reagent for the in situ detection ofnucleic acid sequences comprising one or more DNA sequences which (a)contain the bases adenosine, guanosine, cytosine or thymidine and (b)are complementary to the nucleic acid sequences to be detected, whereinthe DNA sequences include multiple xanthine or lower alkyl substitutedxanthine derivatives labels covalently linked by a linker molecule toany of the bases of the DNA sequences.
 2. A reagent according to claim 1for in situ detection of a chromosome or region of a chromosomecomprising multiple DNA sequences complementary to different portions ofthe chromosome or chromosome region to be detected wherein the labelsare selected from the group consisting of xanthine, 1-methylxanthine,3-methylxanthine, 7-methylxanthine, theophylline, 1,7-dimethylxanthineand caffeine derivatives.
 3. A reagent according to claim 1 for in situdetection of a chromosome or region of a chromosome comprising multipleDNA sequences complementary to different portions of the chromosome orchromosome region to be detected wherein the DNA sequences includemultiple theophylline or caffeine substituted labels covalently linkedthereto.
 4. The reagent according to claim 3 wherein about 1-10% ofbases in the DNA sequences are labeled.
 5. The reagent according toclaim 3 wherein the label is derived fromtheophylline-8-N-(5-hydroxypentylamino)-O-succinoyl-O'-(N'-(3-sulfosuccinimidyl))ester orcaffeine-8-N-(5-hydroxypentylamino)-O-succinoyl-O'-(N'-(3-sulfosuccinimidyl))ester, the number of labels being sufficient to be detected byimmunological techniques while retaining the specific binding propertiesof the DNA with respect to the complementary target sequence orsequences to be detected.
 6. The reagent according to claim 1 whereinthe DNA sequences prior to labeling are obtained from a chromosomal DNAlibrary of the chromosome or chromosome region to be detected.
 7. Thereagent according to claim 6 wherein the DNA sequences have an averagelength of about 300 bp.
 8. The reagent according to claim 1 wherein thelabels are covalently linked to the DNA sequences at aminated cytosinesites.
 9. The reagent according to claim 4 wherein about 5-50% ofcytosine sites in the DNA sequences are aminated by bisulfite catalyzedtransamination with a diaminoalkane having 2 to 6 carbon atoms.
 10. Amethod for making a reagent for in situ detection of a chromosomecomprising:(a) disrupting plasmid DNA containing DNA complementary tothe chromosome or region of chromosome to be detected into fragments,(b) transaminating the DNA fragments, and (c) covalently linking axanthine or lower alkyl substituted xanthine derivative label to thetransaminated DNA fragments.
 11. The method of claim 10 wherein theplasmid DNA is disrupted by sonication into fragments of average lengthof about 300 bp.
 12. The method of claim 10 wherein the DNA fragmentsare transaminated by bisulfate catalyzed transamination withethylenediamine.
 13. The method of claim 10 wherein the label is formedby reaction of DNA withtheophylline-8-N-(5-hydroxypentylamino)-O-succinoyl-O'-(N'-(3-sulfosuccinimidyl))esterorcaffeine-8-N-(5-hydroxypentylamino)-O-succinoyl-O'-(N'-(3-sulfosuccinimidyl))ester.14. A method for preparing a reagent for in situ detection of achromosome or region of a chromosome comprising:(a) transminating anumber of cytosine bases contained in unhybridized DNA sequences havingessentially complementary base sequences with respect to the chromosomeor chromosome region to be detected; and (b) covalently bonding axanthine or lower alkyl substituted xanthine derivatives label to atleast a portion of the transaminated cytosine bases, the portion ofcytosine bases having labels covalently bonded thereto being sufficientto be detected by immunological techniques while essentially retainingthe specific binding properties of the reagent with respect to thechromosome or chromosome region to be detected.
 15. The method of claim14 wherein the cytosine bases are transaminated by bisulfite catalyzedtransamination with ethylenediamine.
 16. A method for the in situdetection of a chromosome or region of a chromosome comprising thefollowing steps in combination:(a) contacting a reagent comprising oneor more DNA sequences which: (i) are complementary to the chromosome orchromosome region to be detected and (ii) include multiple xanthine orlower alkyl substituted xanthine derivatives labels covalently linked tothe DNA sequences, with the chromosome or chromosome region to bedetected under hybridization conditions, and (b) detecting the presenceor absence of the xanthine or lower alkyl substituted xanthinederivative label provided by the reagent hybridized with the chromosomeor chromosome region to be detected.
 17. The method of claim 16 whereinthe reagent is blocked by adding an excess of blocking DNA to thereagent under hybridizing conditions.